Research ArticlePhotothermally Responsive Conjugated Polymeric Singlet OxygenCarrier for Phase Change-Controlled and SustainablePhototherapy for Hypoxic Tumor

Guo Li,1 Ruyi Zhou,1 Weili Zhao,1 Bo Yu,1 Jie Zhou,1 Shujuan Liu,1 Wei Huang,1,2

and Qiang Zhao1

1Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of AdvancedMaterials (IAM), Nanjing University of Posts and Telecommunications (NUPT), 9 Wenyuan Road, Nanjing, 210023 Jiangsu, China2Frontiers Science Center for Flexible Electronics, Xi’an Institute of Flexible Electronics (IFE) and Xi’an Institute of BiomedicalMaterials & Engineering, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China

Correspondence should be addressed to Shujuan Liu; iamsjliu@njupt.edu.cn, Wei Huang; iamdirector@fudan.edu.cn,and Qiang Zhao; iamqzhao@njupt.edu.cn

Received 14 July 2020; Accepted 25 August 2020; Published 10 October 2020

Copyright © 2020 Guo Li et al. Exclusive Licensee Science and Technology Review Publishing House. Distributed under a CreativeCommons Attribution License (CC BY 4.0).

Hypoxia significantly compromises the therapeutic performance of photodynamic therapy (PDT) owing to the oxygen level whichplays a key role in the production of singlet oxygen (1O2). Herein, the photothermally responsive phase change materials (PCM) areused to encapsulate 1,4-dimethylnaphthalene-functionalized platinum(II)-acetylide conjugated polymer (CP1) with intense near-infrared (NIR) absorption to prepare new 1O2 nanocarriers (CP1-NCs). The 1,4-dimethylnaphthalene moieties in CP1-NCs cantrap the 1O2 produced from CP1 under irradiation and form a stable endoperoxide. Then, the endoperoxide undergoescycloreversion to controllably release 1O2 via the NIR light-triggered photothermal effect of CP1 and controllable phase changeof PCM, which can be used for oxygen-independent PDT for hypoxic tumor. Furthermore, the in vivo luminescence imaging-guided synergistic PDT and photothermal therapy showed better efficiency in tumor ablation. The smart design shows thepotent promise of CP1-NCs in PCM-controlled and sustainable phototherapy under tumor hypoxic microenvironment,providing new insights for constructing oxygen-independent precise cancer phototherapeutic platform.

1. Introduction

Photodynamic therapy (PDT), as a well-known and emergingclinical treatment solution, can transform oxygen (O2) intotoxic reactive oxygen species (ROS) to eradicate cancer cells[1–4]. As a consequence, considerable efforts have beendevoted to enhancing PDT due to their minimal invasiveness,high selectivity, accurate spatiotemporal regulation, and mini-mized side effect [5–9]. So far, a largemajority of reported PDTtherapeutic systems depend highly on O2 level [10–14]. How-ever, the consumption of O2 in PDT process and rapid prolif-eration of tumor cells aggravate hypoxic microenvironments[15–17]. Thus, tumor hypoxia is traditionally considered the“Achilles’ heel” of PDT, which not only accelerates metastasisin many solid tumor cells but also significantly causes therapyresistance and low antitumor efficiency [18–23].

To date, various innovative strategies have been exten-sively utilized to relieve tumor hypoxia and enhance the ther-apeutic effect of PDT [18, 20, 21]. One is to integrate PDTwith chemotherapy for synergistic therapy [24–26]. How-ever, such chemophototherapeutic prodrugs are complexmulticomponent nanosystems, which often lead to the diffi-culty in the preparation and suffer from the threat of releaseof drug in normal cells and tissues. Another approach is todirectly transport O2 into tumor sites through oxygen-carrier nanomaterials, including perfluorocarbon, CaO2,MnO2, carbon nitride, enzymes, or oxygenated hemoglobin[27–30]. The enhanced PDT efficiency could be achieved bythe quantitative O2 generation performance. However, inthese oxygen self-supplying nanomaterials, there remainmany severe challenges, including unexpected side effectsand unpredictable toxicity in the complicated physiological

AAASResearchVolume 2020, Article ID 5351848, 14 pageshttps://doi.org/10.34133/2020/5351848

https://doi.org/10.34133/2020/5351848

environment. In addition, many smart phototherapy plat-forms for in situ generation of hydroxyl radicals withintumors based on the unique tumor microenvironment fea-ture through Fenton reaction have been developed, whichpossess high therapeutic specificity and low invasiveness[31–36]. However, most of these nanoplatforms are alsobased on complex multicomponent nanosystems [27–31].Although many prominent works have been developed, thereis an urgent need to construct a new PDT therapeutic systemfor overcoming the tumor hypoxia and achieving sustainablephototherapy.

Herein, we developed a novel photothermally responsiveconjugated polymeric singlet oxygen (1O2) carrier, achievingphase change-controlled and luminescence imaging-guidedsustainable phototherapy for hypoxic tumor (Figure 1). Plati-num(II)-acetylide conjugated polymers (CPs) containingboron dipyrromethene (BDP) units with intense near-infrared (NIR) absorption were chosen as a perfect photo-therapeutic platform [37–44], which simultaneously possessPDT and photothermal therapy (PTT) properties. 1,4-Dimethylnaphthalene, as the efficient 1O2 carrier, was intro-duced into CPs (CP1), which can reversibly trap and photo-thermally release 1O2 (Figure 1(a)), providing a new strategyfor directly delivering 1O2 to relieve hypoxic tumor [45–47].As compared to many other stimulus-responsive platform,photothermally responsive nanoplatform via phase changematerials (PCM) with a large potential fusion heat andreversible solid-liquid phase change in a relative narrow tem-perature region is more appropriate for accurately controlledrelease [48–51]. Therefore, the CP1 nanocarriers (CP1-NCs)were prepared through coencapsulating hydrophobic CP1 inbiocompatible organic PCM (a mixture of oleic acid and hex-adecanol, melting point at 46°C) with amphiphilic lecithinand DSPE-mPEG5000. Meanwhile, the novel CP1-NCs wereinjected into a tumor-bearing mouse via the tail vein andeffectively accumulated in tumor location via the improvedpermeability and retention (EPR) effect [52, 53]. Besides,the CP1-NCs could not only act as a NIR luminescence imag-ing contrast agent but also trap the 1O2 produced from CP1under irradiation and form a stable endoperoxide (EPO).Furthermore, the EPO undergoes cycloreversion to control-lably release 1O2 via the NIR light-induced photothermaleffect of CP1 and controlled phase change of PCM, whichcan be used for oxygen-independent PDT for hypoxictumor. Therefore, the new CP1-NC therapeutic nanoplat-form could successfully relieve tumor hypoxia for oxygen-independent sustainable phototherapy and provide valuableguideline to develop high-performance theranostic agentsin cancer treatment.

2. Results and Discussion

2.1. Synthesis and Characterization. BDP-based CPs (CP1-CP3) were synthesized from the BDP precursor and Pt1 or1,4-diiodobenzene through dehydrohalogenation reaction[54, 55]. The monomer trans-dichlorobis (tri-n-butylpho-sphine) platinum(II) (Pt1) was obtained from the reportedmethods [56–58]. The detailed synthetic routes of BDP pre-cursor and CP1-CP3 are shown in Figures S1 and S2. CP2

and CP3 without Pt center and 1,4-dimethylnaphthalene-functionalized group were designed as the control. CP1-CP3 were purified through silica column chromatographyand precipitated in methanol, generating the dark greenand purple polymers. The 1H NMR, 13C NMR, and matrix-assisted laser desorption/ionization time-of-flight massspectrometry were used to characterize the purity ofintermediates and CPs. Furthermore, the weight averagemolecular mass (Mw) of CP1, CP2, and CP3 was 9500,8500, and 8500 with polydispersity indexes (PDI) of 1.11,1.09, and 1.14, respectively.

As shown in Figure 1(b), the CP1-NC formation wasillustrated. The PCM were prepared according to thereported procedures [48]. Through modulating the massratio of oleic acid (OA) and 1-hexadecanol (Hex) at 1 : 3.5,PCM with a melting point of 46°C were obtained(Figure 2(a)). Through coencapsulating hydrophobic CP1-CP3 into PCM with amphiphilic lecithin and DSPE-mPEG5000, the water-soluble CP1-NC and CP nanoparticles(CP-NPs) were acquired. The concentrations of CP1-NCsand CP-NPs were calculated according to the absorption ofFigure S4. The CP1-NCs were fully characterized bytransmission electron microscopy (TEM) and dynamic lightscattering (DLS). TEM imaging showed that CP1-NCspossessed a uniform morphology, revealing the diameterabout 35 nm (Figure 3(a)). DLS indicated that CP1-NCspossessed an average hydrodynamic size of around 78nm(Figure 3(b)), suggesting the potential passive tumortargeting ability via EPR effect [52].

2.2. Photophysical Properties. The photophysical propertiesof CPs, CP1-NCs, and CP-NPs were explored by theabsorption and photoluminescence (PL) spectra. As exhib-ited in Figures 3(c) and S5, the maximal absorption peaksof CP1-CP3 were located at 660, 575, and 650nm inCH2Cl2, respectively, which are assigned to the π‐π ∗ tran-sition of the BDP platinum-acetylide backbone, showingsignificant red shift in comparison with that of their corre-sponding BDP precursor owing to the π-electron delocali-zation and rigid planar structures [42, 59]. The absorptionspectra of CP1-NCs displayed a strong absorption band at254 nm, which was consistent with the absorption of CP1and 6. After the coordination with BDP precursor 6 or7, CP1-CP3 possessed intense NIR emission in the regionof 650-750 nm (Figure S6). Compared with the CPs, theformed CP-NPs showed significant suppression of theluminescence, which endows a high potential for CP-NPsas a photothermal agent [38, 60]. The strong π‐πstacking in the micellar cores leads to the quenching offluorescence [60]. Furthermore, photostability is the keyin cancer phototherapy, and the resistance of CP1-NCsto photobleaching was measured. The absorption of CP1-NCs showed no significant change during 60min under690 nm light irradiation, demonstrating the excellentantiphotobleaching performance (Figure 3(d)).

2.3. Photodynamic and Photothermal Properties. The photo-thermal effect of CP1-NCs plays an important role in theprocess of triggering 1O2 release. Therefore, the temperature

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elevation of CP1-NCs was firstly explored at various concen-trations under 690nm irradiation (0.5Wcm-2). CP1-NCsshowed remarkable temperature elevation from 21 to 39°Cwith the increasing of concentration, which was much higherthan that of the control CP2-NPs (ΔT ≈ 9°C) (Figures 2(c)and S8). The remarkable temperature elevation of CP1-NCsdemonstrates that the introduction of Pt can enhance thephotothermal efficiency through the improved nonradiativedecay process [38]. Besides, the photothermal conversioneffect of CP1-NCs was tested by temperature elevationthrough a cycle of heat-up and cooling after cessation of lightirradiation. CP1-NCs showed the superior photothermalconversion effect of 49.0% compared with the existing photo-thermal materials such as aza-boron-dipyrromethene dyenanoparticles (43.0%), Pt-based nanoparticles (37.0%), semi-conducting polymer nanoparticles (26.7%), cyanine dyenanocarriers (26.6%), and gold rods (21.0%) [38, 61–64].Moreover, CP1-NCs exhibited almost no large difference in

their temperature monitoring after five irradiation/coolingcycles (Figure S8(a)), suggesting their superior resistance tophotobleaching under irradiation.

Then, we explored the morphological changes of the CP-NCs before and after 690 nm laser irradiation. TEM imagingsuggested that light irradiation generated significant influ-ence not only on the particle size but also on the morphologyof the CP1-NCs@PCM (Figures 2(d) and 2(e)). After irradi-ation, the morphological change of CP1-NCs was observedfrom uniform sphere to amorphous structure by TEM imag-ing. And the hydrodynamic diameters of the nanocarriersbecame smaller (from 74 ± 6 nm to 36 ± 5 nm) through DLS(Figure S3). For CP2-NPs, no significant difference wasfound in the particle size and morphology of the CP2-NPs@PCM after irradiation, which was attributed to thenegligible photothermal efficiency of CP2-NPs.

Afterwards, the sustainable PDT property of CP1-NCswas demonstrated through investigating the 1O2 trap and

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modified nanoprecipitation and the photothermal-triggered release of CP1-NCs and PCM. (c) The responsive process of controlled andsustainable phototherapy under hypoxia.

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Figure 2: (a) Melting point change of PCM with different Hex/OA mass ratios. (b) Photothermal images of CP1-NCs. (c) Temperatureelevation of CP1-NCs at different levels under 690 nm light irradiation at 0.5W cm-2 for 6min. TEM of CP1-NCs, (d) before, and (e) afterirradiation for 6min. Scale bars: 200 nm. (f) ΔAbs of DPBF under various temperatures in the mixture solution of CP1-NCs and DBPF.(g) Schematic illustration of photothermal-induced release of CP1-NCs.

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release process. The 1O2 capture capability of CP1-NCs wasinvestigated through the absorption decrease due to theEPO formation. Along with the increase of irradiation time,the absorption band at 254nm decreased gradually, whichdemonstrated that 1O2 sensitized by CP1-NCs was capturedby itself to generate EPO (Figure S7). Under 690 nmirradiation, the CP1-NCs were able to sensitize oxygen toproduce 1O2, which displayed a decreasing in absorbance at420nm (ΔAbs) in the presence of 1O2 by 1,3-diphenylisobenzofuran (DPBF) as an indicator (Figure S7(a)).Furthermore, the 1O2 release of CP1-NCs was carried outunder dark conditions at three temperatures of 37, 45, and60°C, respectively. As exhibited in Figure 2(f), a highertemperature could accelerate the release of 1O2. In contrast,when 1,4-dimethylnaphthalene-free CP3-NPs were irradiated,the negligible ΔAbs values were observed (Figure S7(b)). Theresults suggested that CP1-NCs not only could effectivelyform EPO under irradiation but also release 1O2 by thephotothermal effect in aqueous solution. As shown inFigure 2(g), a hypothetical model further illustratedphotothermal-induced release of CP1-NC under irradiation.Especially, compared to traditional photosensitizer, O2 isunnecessary in 1O2 release process of CP1-NCs, suggestinggreat potential for enhancing the phototherapeuticperformances of hypoxia-associative PDT. Therefore, theexcellent PDT/PTT performances of CP1-NCs endowed it aspotential phototherapy agents for the following NIR light-induced therapy experiments.

2.4. Cytotoxicity Assay and PDT/PTT Efficiency of CP1-NCs.The cytotoxicity of CP1-NCs in vitro was explored throughthe 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-lium (MTT) assessment. HeLa cells were treated with CP1-NCs at various concentrations (1.0, 2.0, 4.0, 6.0, and8.0μgmL-1) for 24h and then conducted with 690nm lightirradiation under 21% and 5% O2 for 6min or not, respec-tively (Figure 4(a)). The HeLa cell viability in the absence ofirradiation was larger than 77% at the level of 8.0μgmL-1,demonstrating the outstanding biocompatibility of CP1-NCs. Furthermore, the cells cultured with 1O2 loading CP1-NCs displayed relatively lower cell viability under 21% or5% O2 concentration, showing that the CP1-NCs could trig-ger cell oxidative damage and kill cells even under hypoxiacondition. Owing to the intrinsic NIR emission of CP1-NCs, the cellular uptake of CP1-NCs was further explored.The red fluorescence in the cytoplasm was found from thecells cultured with CP1-NCs through the confocal images,indicating the abundant uptake and excellent dispersion ofCP1-NCs in HeLa cells (Figure S9). To investigate thephototherapy performance of the CP1-NCs including 1O2generation, 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) [65], which could be transformed into DCFwith green luminescence in the presence of 1O2, was used asa 1O2 indicator. As exhibited in Figure 4(b), the strong greenluminescence was observed from the HeLa cells treated withCP1-NCs under 21% and 5% O2 level, illustrating theproduction of abundant intracellular 1O2 under irradiation.Moreover, the CP1-NCs generated negligible fluorescence inthe absence of irradiation, since the physiological

temperature (around 37°C) was rather low so that it mightnot induce the release of 1O2 from EPO. The resultssuggested that the release of 1O2 from EPO might betriggered through the photothermal effect of CP1-NCs.

To explore the in vitro cell killing performance, calceinAM (live cells) and propidium iodide (dead cells) stainingassessments were carried out under normoxia and hypoxia.When treated with CP1-NCs, the cells were dead undereither 21% or 5% oxygen level with irradiation(Figure 4(c)). In the absence of light irradiation, the HeLacells incubated with CP1-NCs mainly maintained alive under21% and 5% oxygen levels. The results confirmed that thephototherapeutic performance was unaffected by the hyp-oxia. In addition, to demonstrate the cell population at differ-ent periods of apoptosis, flow cytometry assay was explored(Figures 4(d) and S10). Under 690nm light irradiation, thesignificant improved cells were achieved in the fluoresceinisothiocyanate- (FITC-) positive and PI-positive area undernormoxia and hypoxia. The results demonstrated that thephotothermal efficiency of CP1-NCs induced the release of1O2 and oxidative damage improved the phototherapeuticperformances, motivating us to explore the application ofCP1-NCs for enhanced tumor phototherapy.

2.5. In Vivo Luminescence Imaging, Photothermal Imaging,and DCFH-DA Staining of CP1-NCs. According to the NIRemission of CP1-NCs, the in vivo luminescence imaging ofCP1-NCs was used to acquire the accurate therapeutic time.The CP1-NCs were intravenously injected into the tumor-bearing mice, and their biodistributions were recorded at var-ious postinjection time points. The luminescence signal inthe tumor sites quickly increased from 2h to 12h after injec-tion and decreased as the metabolism of CP1-NCs(Figure 5(a)). The signal intensity reached to its plateau at12 h postinjection, which was acted as the reference timefor the following therapy. In comparison, negligible or weakluminescence intensity was showed in other main organs(Figure S11). These imaging results suggested superiorability of the CP1-NCs to accumulate in tumor location,which was attributed to the EPR effect via the properparticle size.

To explore the performance of CP1-NCs to producehyperthermia in vivo, CP1-NCs were injected into the miceat different doses, followed through infrared thermal imagingof mice after 12 h injection under 690 nm irradiation(0.5Wcm-2). PBS, as a control, led to a negligible tempera-ture increase (Figure 5(b)). Under 690 nm irradiation, CP1-NCs at the doses of 0.45, 0.9, and 1.8mgkg-1 CP1 triggeredthe tumor temperature changes of 11, 19, and 30°C, respec-tively, suggesting remarkable temperature elevations withthe increasing of dose (Figures 5(b)–5(d)). Because too hightemperature increase produced detrimental influence forthe normal cell, and too low temperature cannot obtainappropriate photothermal therapy efficiency, the level ofCP1-NCs at 0.9mgkg-1 CP1 was selected for the subsequenttumor therapy. Furthermore, the capability of CP1-NCs pro-ducing ROS at the tumor slice was investigated by theDCFH-DA staining under 690 nm light irradiation. VitaminC (VC) as a 1O2 scavenger was used to scavenge

1O2. As

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displayed in Figure 5(e), the strong green luminescencewas found at the tumor slice of mice cultured with CP1-NCs under light irradiation. However, the HeLa cells withCP1-NCs without light irradiation or in the presence ofVC induced negligible green luminescence. The resultssuggested that CP1-NCs could generate abundant 1O2under irradiation.

2.6. Synergistic Phototherapy. Encouraged by the preferableluminescence imaging, sustainable 1O2 production, and goodphotothermal conversion efficiency of CP1-NCs, the photo-therapy effect of CP1-NCs was carried out in vivo using theHeLa tumor mouse model. After the tumor volume increasedaround 120mm3, the mice were separated into four groups.The mice were treated with (I) PBS, (II) CP1-NCs only,

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(III) CP1-NCs (150μL, 0.9mgkg-1 CP1)+irradiation(690 nm, 0.5Wcm-2)+VC, and (IV) CP1-NCs (150μL,0.9mgkg-1 CP1)+irradiation (690 nm, 0.5Wcm-2), respec-tively. The fourth group acted as the therapy group. The I-III groups served as control. The mouse tumor volume andweight were recorded every two days. For groups (III) and(IV), 6min irradiation was implemented after 12 h injection.As exhibited in Figure 6(a), the tumor volumes of (I) and (II)exhibited an expeditious growth in the absence of irradiation,demonstrating the excellent biocompatibility of CP1-NCs.

Compared with tumors treated with CP1-NCs+irradiation+VC, the tumor was significantly inhibited for those treatedwith CP1-NCs+irradiation, suggesting that the continuousdelivery of 1O2 leads to better phototherapy performance.Similar body weight change was observed in the controlgroups in comparison with the treatment group, suggestingthe negligible side effects of CP1-NCs (Figure 6(b)). Thephoto of tumors was consistent with the monitored tumorweight (Figures 6(c) and 6(d)). The therapy results confirmedthat CP1-NCs can achieve enhanced PDT performance

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Figure 5: In vivo photothermal performances and production of singlet oxygen of CP1-NCs. (a) Luminescence image of the HeLa tumor-bearing mice at various time points through tail intravenous injection of CP1-NCs (n = 3 mice per group). (b) Infrared thermograph ofthe mice bearing HeLa tumor treated with CP1-NCs at different concentrations under 690 nm irradiation at 0.5W cm-2 for 6min and (c)the corresponding temperature elevation within tumor region. (d) Representative thermal images of mice (tumor sites) subjected to690 nm light irradiation for 12 h postinjection of CP1-NCs (0.9mg kg-1, 120μL) and PBS. (e) DCFH-DA staining of tumor slice from themice treated with CP1-NCs at 12 h postinjection in the absence or presence of VC under light irradiation or not (scale bar: 100 μm).

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through the 1O2 release for inhibiting tumor growth, whichcould act as a potential phototherapy agent for precisiontumor ablation. Furthermore, to explore the dark toxicity of

CP1-NCs, the hematoxylin and eosin (H&E) analysis of thetumor and major organs (heart, liver, spleen, lung, and kid-ney) acquired from the mice was measured after therapy.

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The pathomorphology analysis indicated similar morphologi-cal properties with negligible cell and tissue damage in themajor organs for all mice (Figure 6(e)). The results validatedthat CP1-NCs could not generate significant photodamagefor normal organs, although they might accumulated there.

To explore the biological toxicity of CP1-NCs, blood rou-tine analysis and blood biochemistry were carried out. Nosignificant changes of the blood biochemistry (Figure S12)and blood hematology (Figure 7) during the phototherapyprocess were found, suggesting that the CP1-NCs possessexcellent biocompatibility.

3. Conclusion

In summary, we reported a new photothermally responsiveconjugated polymeric 1O2 carrier for phase change-controlled and luminescence imaging-guided sustainablephototherapy for hypoxic tumor. The CP1-NCs not onlycan serve as NIR luminescence imaging agent for real-timeidentification of the nanocarrier accumulation but also cantrap the 1O2 produced from CP1 under irradiation and forma stable EPO. The EPO undergoes cycloreversion to release1O2 through the NIR-triggered photothermal efficiency ofCP1 and controlled phase change of PCM, which can be usedfor oxygen-independent PDT for relieving tumor hypoxicmicroenvironment. As compared with CP1-NCs plus VC,the CP1-NCs exhibit better efficiency in inhibiting tumorgrowth through 1O2 release for enhanced cancer photother-

apy. Therefore, the present study demonstrates the potentialof CP1-NCs in controlled and sustainable cancer photother-apy under hypoxia, affording a new strategy for developingoxygen-independent cancer phototherapy platform.

4. Materials and Methods

4.1. Materials. Unless otherwise stated, all raw materials werebought and utilized as received. Oleic acid and 1-hexadecanolwere obtained from Aldrich chemistry. The biocompatible α-lecithin and amphiphilic DSPE-mPEG5000 were purchasedfrom Adamas-Beta. Calcein AM/PI stain kit was obtained fromNanjing KeyGen Biotech Co., Ltd. The detailed synthesis ofintermediates andCPs can be found in the Supporting Informa-tion. Oleic acid and 1-hexadecanol were weighed at the variousmass ratios (OA : Hex = 1 : 2:5, 3:0, 3:5, 4:0, and 4:5) andthen dissolved in ethanol. Then, the mixture solution was ultra-sonically mixed and stored in a refrigerator as PCM withcontrollable melting point.

4.2. Preparation and Characterization of CP-NPs. The CP-NPs were obtained through a resolidification method. TheCPs and PCM (2mg/mL) dissolved in THF solution serveas solution 1. The DSPE-mPEG5000 and α-lecithin (10mgin 10mL water) dispersed in aqueous solution were used assolution 2. Then, the two solutions were mixed under contin-uous sonication at 50°C for 6min and then rapidly cooled inan ice bath. After the solution was warmed up to room

1 d 7 d 15 d0.0

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NO

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/L)

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(109

/L)

1 d 7 d 15 d

1 d 7 d 15 d0

10

20

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50

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(%)

0.0

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(%)

1 d 7 d 15 d

1 d 7 d 15 d0

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(fL)

0

2

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(109

/L)

1 d 7 d 15 d

0

2

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6

8

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(fL)

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C (g

/L)

1 d 7 d 15 d0

30

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HG

B (g

/L)

1 d 7 d 15 d0

2

4

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LY (1

09/L

)

1 d 7 d 15 d0

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20M

CH (p

g)

1 d 7 d 15 d0

2

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RBC

(101

2 /L)

ControlCP1-NCs

Figure 7: Blood hematology analysis from healthy and CP1-NC-treated mice performed at 1, 7, and 15 days. The hematology indicatorsinclude white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), meancorpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), plateletcrit (PCT), lymphocyte(LY), mean platelet volume (MPV), and monocyte count (MONO). Error bars correspond to standard deviation for n = 3.

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temperature, THF was removed under stirring. The solutionwas filtered via a polyethersulfone (PES) syringe-driven filter(0.44μm) and was further centrifuged utilizing a centrifugalfilter. The concentration of CP-NPs was determined accord-ing to absorption spectra. The morphology and particle sizeof CP-NPs were measured by transmission electron micros-copy and dynamic light scattering.

4.3. Singlet Oxygen Test. To explore the singlet oxygen pro-duction and photodynamic improvement of CP-NPs, DPBFserved as an 1O2 indicator. CP-NPs at different concentra-tions were mixed with DPBF in water solution under contin-uous stirring. Under 690nm irradiation, the absorptionspectra of DPBF were acquired.

4.4. Photothermal Performance. The temperature elevation ofCP-NCs at different concentrations (75, 150, and 300μgmL-1) under 690nm irradiation (0.5Wcm-2, 6min) was recordedby the FLIR E40. To acquire the photothermal conversionefficiency, CP1-NCs were exposed under 690nm light irradi-ation at 0.5Wcm-2. After the temperature reached a plateau,the light irradiation was removed for cooling down to ambi-ent temperature.

4.5. Cellular Experiment. The cells were treated in Dulbecco’smodified Eagle medium and provided with 10% fetal bovineserum (FBS) at 37°C with 5% CO2. To evaluate the cellularuptake, HeLa cells were incubated with CP1-NCs in the darkfor 6 h. Then, the cells were washed with fresh PBS twicebefore being imaged by confocal imaging. To test the photo-toxicity of CP1-NCs, HeLa cells were treated with PBS orCP1-NCs under 21% or 5% oxygen concentration for 24h,followed by 5min light irradiation under 690nm(0.5Wcm-2). The HeLa cells were incubated with DCFH-DA at 37°C under 21% or 5% oxygen level for 30min,followed by 690 nm irradiation at 0.5Wcm-2 for 5min. Fur-thermore, to explore the cell population at different stages ofapoptosis, AM/PI and flow cytometry assay was carried outunder normoxia and hypoxia.

4.6. In Vivo Fluorescence Imaging. For in vivo luminescenceimaging, HeLa tumor-bearing mice were intravenouslyinjected with CP1-NCs. The luminescence signals of the micewere collected (λex = 690 nm) through an IVIS Lumina Kin vivo imaging system (PerkinElmer) using a xenon lampwhich was equipped with different long- and band-pass fil-ters. An Andor EMCCD-DU897 camera was used as animaging detector. Images were taken at 2, 4, 8, 12, and 24 hpostinjection. After 24h, the tumor and major organs werecollected and the fluorescence intensity was analyzed to con-firm the accurate therapeutic time.

4.7. Blood Routine Examination. The whole blood wasacquired from the orbital venous plexus on 1-, 7-, and 15-day postinjection and subject to blood routine test. The bloodsamples were acquired from the mouse fundus artery in eachgroup. Blood solutions (100μL) were treated with anticoagu-lant for hematology analysis. After being kept at 4°C for 4 hand centrifuged, blood plasma samples (200μL) wereobtained from blood for biochemistry assay.

4.8. In Vivo Synergistic Therapy. All tumor nude mice wereacquired from Jiangsu KeyGen Biotech Co., Ltd. and usedreferring to the standard of the Laboratory Animal Centerof Jiangsu KeyGen Biotech Co., Ltd. When the tumor volumeincreased to around 120mm3, we began to carry out the pho-totherapy experiments. To assess the phototherapy effect ofCP1-NCs, the tumor-bearing HeLa mice were injected withCP1-NCs at a dose of 0.9mgkg-1. The tumors were exposedunder 690nm light irradiation at 0.5Wcm-2 for 6min.

4.9. Histological Staining. After treatment, the final tumorand major organs were fixed with 4% formaldehyde forH&E staining to explore the side effect of CP1-NCs.

Conflicts of Interest

The authors declare no competing financial interests.

Authors’ Contributions

Guo Li, Qiang Zhao, Shujuan Liu, and Wei Huang designedthe project. Guo Li carried out the material synthesis andcharacterizations. Guo Li, Shujuan Liu, and Qiang Zhao ana-lyzed the whole project as well as wrote the manuscript. RuyiZhou and Bo Yu performed the in vitro experiments. WeiliZhao and Jie Zhou carried out the cell measurement. Allthe authors were included in the discussion and approvedthe manuscript.

Acknowledgments

This work was supported by the China National Funds forDistinguished Young Scientists (61825503), the National Nat-ural Science Foundation of China (61775101 and 61805122),the National Key Research and Development Program ofChina (2017YFA0205302), and the Open Research Fund ofState Key Laboratory of Bioelectronics, Southeast University.

Supplementary Materials

Figure S1: synthetic routes of the 1-7. Figure S2: syntheticroutes of the CP1-CP3. Figure S3: morphological changesof the CP-NCs before and after NIR irradiation. The size ofthe CP1-NCs@PCM became smaller after the irradiation,while no obvious changes were obtained from the CP2-NPs@PCM. Scale bars: 200 nm. Figure S4: (a) UV-vis-NIRabsorption spectra of CP1 at various concentrations inCH2Cl2. (b) The equation was calculated according to themaximal absorption of (a). (c) UV-vis-NIR absorption spec-tra of CP2 at various concentrations in CH2Cl2. (d) Theequation was calculated according to the maximal absorptionof (c). (e) UV-vis-NIR absorption spectra of CP3 at variousconcentrations in CH2Cl2. (f) The equation was calculatedaccording to the maximal absorption of (e). Figure S5:absorption spectra of 7, CPs, and CP-NPs. Figure S6: emis-sion spectra of CP1-CP3 in CH2Cl2 and CP-NPs in water.Figure S7: (a) absorption changes of DPBF with CP1-NCsunder 690nm irradiation in water and (inset) the absorptiondecrease owing to the endoperoxide formation. (b) ΔAbs ofDPBF in mixture solution of CP3-NPs and DBPF. Figure

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S8: (a) photothermal stability of CP1-NCs. (b) The heatingcurve of the CP1-NCs in a procedure of laser-on and laser-off. (c) The linear cooling time data versus −ln ðθÞ acquiredfrom the cooling period of (b). (d) Temperature elevationof CP2-NPs. Figure S9: confocal images of the cellular uptakeof CP1-NCs and DAPI. Scale bar: 20nm. Figure S10: flowcytometry quantification of annexin V-FITC and PI-labeledHeLa cells cultured with only PBS or laser, respectively. FigureS11: the CP1-NC distribution in the tumor and major organsafter 12h intravenous injection. Figure S12: blood biochemicalassay. (a) Glutamic-pyruvic transaminase (ALT), (b) glutamicoxalacetic transaminase (AST), (c) urea nitrogen (BUN), and(d) creatinine (CREA) concentrations for liver and kidneyfunctions of healthy nude mice 15 days after tail intravenousinjection of CP1-NCs. Error bars represent the standard devi-ations (n = 3). Figure S13: the 1H NMR spectrum of 6 inCDCl3. Figure S14: the

13C NMR spectrum of 6 in CDCl3. Fig-ure S15: the 1H NMR spectrum of 7 in CDCl3. Figure S16: the1H NMR spectra of CP1-CP3. Figure S17: MALDI-TOF-MSspectra of 4-6. (Supplementary Materials)

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Photothermally Responsive Conjugated Polymeric Singlet Oxygen Carrier for Phase Change-Controlled and Sustainable Phototherapy for Hypoxic Tumor1. Introduction2. Results and Discussion2.1. Synthesis and Characterization2.2. Photophysical Properties2.3. Photodynamic and Photothermal Properties2.4. Cytotoxicity Assay and PDT/PTT Efficiency of CP1-NCs2.5. In Vivo Luminescence Imaging, Photothermal Imaging, and DCFH-DA Staining of CP1-NCs2.6. Synergistic Phototherapy

3. Conclusion4. Materials and Methods4.1. Materials4.2. Preparation and Characterization of CP-NPs4.3. Singlet Oxygen Test4.4. Photothermal Performance4.5. Cellular Experiment4.6. In Vivo Fluorescence Imaging4.7. Blood Routine Examination4.8. In Vivo Synergistic Therapy4.9. Histological Staining

Conflicts of InterestAuthors’ ContributionsAcknowledgmentsSupplementary Materials